Fluorescence-activated cell sorting and directed evolution of α-N-acetylgalactosaminidases using a quenched activity-based probe (qABP)

Article abstract of DOI:10.1039/c3cc42836b

An α-N-acetylgalactosamine-containing quenched activity-based probe (qABP) was designed and successfully synthesized, and it was subsequently used in directed enzyme evolution experiments aided by fluorescence-activated cell sorting (FACS) for the discove

Full text of DOI:10.1039/c3cc42836b

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Fluorescence-activated cell sorting and directed
evolution of a-N-acetylgalactosaminidases using a
quenched activity-based probe (qABP)†
Cite this: Chem. Commun., 2013,
49, 7237
Received 17th April 2013,
Accepted 19th June 2013
a
b
b
Kamaladasan Kalidasan,z Ying Su,z Xiaoyuan Wu,z Shao Q. Yao*b and
Mahesh Uttamchandani*abc
DOI: 10.1039/c3cc42836b
www.rsc.org/chemcomm
An a-N-acetylgalactosamine-containing quenched activity-based probe image target enzymes, specifically caspases and phosphatases, within
(qABP) was designed and successfully synthesized, and it was subse- precise sub-cellular locations of mammalian cells.11 While Kwan et al.
quently used in directed enzyme evolution experiments aided have similarly applied quinone methide-based probes to isolate cells
by fluorescence-activated cell sorting (FACS) for the discovery of an expressing or not expressing certain glycosidases,12 we demonstrate
a-N-acetylgalactosaminidase variant with improved catalytic activity.
here, for the first time, the utility of such probes for evolving galacto-
sidase activities, using error-prone PCR and rounds of FACS selection
Fluorescence-activated cell sorting (FACS), when combined with of mutant libraries, in E. coli (Fig. 1A).
random mutagenesis, offers a potent strategy for directed protein
We elected to apply this strategy to a unique a-N-acetyl-galactos-
evolution.1–3 At the heart of this technique are the range of proteins, aminidase (NAG), recently discovered from a soil bacterium Eliza-
dyes or probes which transduce the intended biological event into bethkingea miricola.13 This enzyme was found to selectively hydrolyse
quantifiable fluorescent signals in cells.4,5 Both fluorogenic substrates the a-N-acetyl-galactosamine (A-antigen) of red blood cells (RBCs)
and activity-based probes (ABPs) may conceivably be used to drive surface glycosides, with potential applications in converting ‘‘A’’ type
selection using FACS, enabling potential high-throughput identifi- blood stocks to the universal ‘‘O’’ type blood (Fig. 1B).14,15 At current
cation and separation of cell populations, which express desirably activities, sixty milligrams of the wildtype NAG enzyme is required to
active protein variants.6,7 Current strategies are however caught
between two extremes: fluorogenic probes upon activation easily
diffuse out of cells, diminishing the positive fluorescent readout
needed for FACS selection.8,9 Conversely, ABPs designed as suicide
inhibitors are persistently fluorescent (always on, resulting in higher
backgrounds/false positives) and bind to the enzyme active site
covalently, limiting the catalytic turnover and positive readout.4,10
In order to address these limitations, we herein report the design of
a ‘smart’ quinone methide-based quenched activity-based probe
(qABP) with the following special features: (1) a modular structure, (2)
turn-on fluorescence, (3) ability to diffuse away from the enzyme active
site (facilitating multiple turnovers froma single enzyme molecule) and
(4) retention within the cell (thus magnifying fluorescent signals in
target cell populations). We have previously applied our probe design to
a Defence Medical and Environmental Research Institute, DSO National
Laboratories, 27 Medical Drive, Singapore 117510, Singapore
b Department of Chemistry, National University of Singapore, 3 Science Drive 3,
Singapore 117543, Singapore. E-mail: chmyaosq@nus.edu.sg; Fax: +65 6779 1691;
Tel: +65 6516 2925
c Department of Biological Science, National University of Singapore,
14 Science Drive 4, Singapore 117543, Singapore. E-mail: mahesh@dso.org.sg;
Fig. 1 (A) Overall strategy of evolving enzymes using a smart qABP and FACS.
The mutant library is sorted and subjected to FACS. (B) Structure of glycolipids on
the surface of Type A red blood cells, which upon cleavage by NAG at the position
indicated with a dotted line would produce Type O RBCs. (C) Structure of Probe A
and mechanism of action. W: warhead; F: fluorophore; Q: quencher.
Fax: +65 6485 7033; Tel: +65 6485 7214
† Electronic supplementary information (ESI) available: Synthesis scheme and
characterization of intermediates, microplate/gel results and FACS optimization.
See DOI: 10.1039/c3cc42836b
‡ Joint first authors.
c
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Scheme 1 Probe synthesis strategy. Fluorescein was selected as the fluorophore and dabcyl as the quencher.
convert one pint of packed RBCs (200 ml volume) under a 1 h
incubation.13 At these quantities, scaling up can be prohibitively coumarin Aa probes in vitro (Fig. S1A, ESI†). As expected, these
costly, hence there is a need for more efficient enzymes. probes provided high fluorescent readouts, confirming the
We initially tested the purified NAG enzyme with commercial
Probe A was designed with an a-N-acetyl-galactosamine war- expected enzymatic activity (Fig. S1B, ESI†). When tested with
head (W), a fluorescence reporter fluorescein (F) and a dabcyl Probe A, pure NAG also exhibited strong enzyme activity-
quencher (Q), built around a mandelic acid core (Fig. 1C). Upon dependent fluorescent readouts, establishing compatibility with
intracellular cleavage by NAG to release the warhead, a 1,6- our probes in vitro (Fig. 2B and Fig. S1C, ESI†). NAG-expressing
elimination reaction would liberate the dabcyl module, generat- cell lysates also produced positive readouts (Fig. S1, ESI†). The
ing fluorescence and a reactive quinone methide intermediate probes were also tested against the NovaBlue(DE3) lysates,
that would subsequently covalently react with nearby intracellu- confirming no background activity. By running the probe-labeled
lar nucleophiles (Fig. 1C). Such probes are ill-suited as ABPs for lysates on the gel followed by in-gel fluorescence scanning, it was
target identification as they diffuse away from the enzyme active revealed that a broad spectrum of endogenous proteins was
site. Nevertheless, for bioimaging of localized intracellular labeled by Probe A in the presence of active NAG (Fig. S2, ESI†),
enzyme activities as previously demonstrated,11 as well as direc- indicating successful intracellular trapping of the probe.
ted evolution using FACS, which is reported in the current study,
Probe delivery into E. coli cells was vital for cell-based screening of
this is exactly the property that we intended to exploit, to retain enzyme activity during FACS analysis, and we needed to establish an
enzymatic activity while magnifying differences across variants. effective cell-loading protocol for the probe. The performance of
Probe A was synthesized over 11 solution-phase steps loading was measured using FACS readouts with NAG-overexpressing
(Scheme 1 and Scheme S1, ESI†). Briefly, the commercial galactos- NovaBlue(DE3) cells (Fig. 3A). Upon optimization (see ESI†), we were
amine 1 was converted to the corresponding azide and reacted able to find conditions under which a normally distributed
with acetic anhydride, giving 2. The hydroxide at position 1
was selectively deprotected, giving 3. Upon introduction of the
mandelic acid core, the corresponding azide intermediate 5 was
obtained. Direct conversion of N3 in 5 to NHAc was achieved in a
single-step, giving 6. Next, dabcyl-NH2 was introduced, producing
8. Finally, fluorescein was introduced to produce 10, which upon
further deprotection afforded Probe A.
Next, NAG was cloned into pDEST17 using the Gateway
cloning system (Invitrogen) (Table S1, ESI†). The library was
generated using error-prone PCR (see ESI†) which was then
transformed into NovaBlue(DE3) competent cells by electro-
poration. These cells were well suited as their LacZ (b-galacto-
sidase) gene was disrupted. They also possessed a T7 promoter
Fig. 2 (A) Purified NAG derived from Elizabethkingia miricola. Lanes – MW: mole-
cular weight marker (sizes in kD); (1) uninduced lysates; (2) induced lysates; (3) purified
NAG protein (expected MW 46 kD). (B) Concentration-dependent activity of WT NAG
system compatible with pDEST17. The purified wildtype NAG
enzyme was well-expressed and active (Fig. 2).
against 20 mM Probe A in vitro. Red curve ꢀ 20 nM NAG; green curve ꢀ 2 nM NAG;
blue curve – blank, with probe only. Readouts obtained at l490 nm/520 nm
.
c
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proceeded to select novel NAG variants with enhanced activity. A
further top 0.2% of cells were sorted in the second round. After
each round, half of the sorted samples were plated while the
other half were grown for another round of sorting. Individual
colonies were randomly sequenced colonies from each round of
sorting (representative results in Table S3, ESI†).
The NAG enzyme is grouped within the GH109 family of glycosi-
dases. Its structure has been established, revealing residues Tyr-307,
Tyr-225, His-228 and Glu-149 are involved with substrate binding in
the enzyme active site.13 Based on the sequences and structural
positions of mutation sequences, we selected five Round-2 colonies
with mutations near the active site for follow-up (Table S3, ESI†).
However, we could only successfully purify the enzyme from one of the
colonies and hence quantified its activity profiles carefully. The yields
from the other variants were extremely low, when purification was
attempted under native conditions. The purified variant carried two
non-synonymous mutations (H104L and S347R) (Table S2 and Fig. S3,
ESI†). Both these mutations are located within 20 Å of the enzyme
active site (Fig. 4C). This variant NAG was found to exhibit a lower KM
and close to two times higher kcat/KM ratio than the wildtype NAG
(Fig. 4). This correlated well with expectations from the FACS experi-
ments. No further variants with enhanced activities were identified.
In conclusion, we evolved a sub-optimal enzyme to a more
active form using the smart qABP strategy developed herein.
Compared to traditional fluorogenic probes, quinone methide
qABPs do not diffuse out of cells readily, so continued measure-
ments of the fluorescence signals will be possible. As far as we
know, this is the first example of using such smart quinone
methide-based probes for evolving enzyme activity. The novel
NAG variant identified will be tested on human RBCs to
evaluate its efficacy. We anticipate that this strategy may be
applied more broadly in enzyme engineering and evolution.
The authors acknowledge funding support from DSO
National Laboratories, Singapore (Grant no. 20090211). Ms
Tan Pei Xin developed the NAG constructs used in this study.
Fig. 3 FACS sorting results. (A) Wildtype NAG expressing cells sorted with and
without Probe A (red and blue histograms respectively). (B) Mutant library sorted
with and without probe (red and blue histograms respectively). (C) The bottom
0.5% and the top 0.2% library populations were re-grown and sorted, generat-
ing the distinct green and orange histograms respectively.
population of positively fluorescent cells was obtained consistently. A
significant proportion of the cell population generated fluorescence
readouts above 100 RFUs, with greater than 95% of the cell popula-
tion producing consistent readouts greater than 10 RFUs. By incu-
bating the cell population for extended period of times on ice (to
limit cell division), we found that the cells produced nearly identical
sorting profiles, and the probe was successfully retained in the cells
for at least several hours without significant loss of fluorescence.
Thereafter, we tested the library of NAG variants with Probe A.
We found that a large fraction of electroporated mutant library
cells incubated with Probe A emitted fluorescence (Fig. 3B, red
curve), when compared to the group that contained
Novablue(DE3) cells without Probe A (Fig. 3B, blue curve). In
order to test that we could select desired populations of active
variants, we proceeded to sort out the top 0.2% of the electro-
porated mutant library. We also sorted the bottom 0.5% of the
library. These two populations were then re-grown in culture and
studied using FACS (Fig. 3B, orange and green curves respec-
tively). The results showed that there was a marked difference in
the activity of the sorted cell populations, indicating that higher-
activity variants could be enriched using the strategy. We thus
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Fig. 4 (A) Kinetic activities of the control and the variant NAG enzymes (H104L,
S347R) were plotted on a Michaelis–Menten plot (averaged from triplicates). (B)
KM and kcat values of the NAG wildtype and variant were calculated by non-linear
regression, least squares fitting, ꢁ95% confidence intervals. (C) The wildtype NAG
enzyme is shown on the left. The variant NAG enzyme (with protein ribbon in red)
has mutated amino acids H104L, S347R in black. The active site residues are shown
in blue. Figures are adapted from PDB entry 2IXA13 and rendered in Pymol.
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c
This journal is The Royal Society of Chemistry 2013
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